UA97233C2 - Process for manufacturing vaccines - Google Patents

Abstract

Disclosed is a process for making the immunogenic composition by conducting saccharide-protein conjugation reactions using carbodiimide condensation chemistry. Depending on the nature of the saccharide or protein carrier involved, the quality of the conjugate may be improved by adding one of the reaction components slowly to the reaction mixture. Further provided are imunogenic compositions comprising the saccharide-protein conjugates made by the methods disclosed.

Description

a) mixing of the saccharide and an aliquot of carbodiimide required for conjugation, and p) adding an aliquot of the carrier protein is carried out within a period of 35 seconds to b hours; 11) - if the saccharide contains both amino and carboxyl groups and the carrier protein contains either amino or carboxyl groups: a) mixing the carrier protein and an aliquot of carbodiimide required for conjugation, and p) adding an aliquot of the saccharide is carried out during the period from 35 seconds to 6 hours;

I) - if the carrier protein contains both amino and carboxyl groups and the saccharide contains both amino and carboxyl groups: a) mixing the carrier protein and saccharide, and p) adding an aliquot of carbodiimide necessary for conjugation is carried out during period from seconds to b hours.

Any suitable carbodiimide can be used provided it is capable of condensing saccharides and proteins in an aqueous medium. In one of the embodiments of the present invention, the carbodiimide can be EVAS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (also known as EOS| or it can be another carbodiimide than ECAS.

The term "saccharide" throughout the text of the description can mean a polysaccharide or an oligosaccharide and includes both meanings. It can also mean lipopolysaccharide (II RB5) or lipooligosaccharide (GO5). Before use, polysaccharides (such as bacterial polysaccharides) may be isolated from a source strain (e.g., bacteria) or isolated from a source strain and fractionated to some degree by known methods (see, e.g.,

EP497524 and EP497525; Zpoizip Spep 521 her ai. - Sagropuagaiye Nezeeaagsp My! 152 p7-20 (1986)) for example, by microfluidization. Polysaccharides can be fractionated in order to lower the viscosity of the polysaccharide samples and/or improve the filterability of the conjugated products. Oligosaccharides have a low number of repeating fragments (typically 5-30 repeating fragments) and are typical hydrolyzed polysaccharides.

The meaning of the term "carrier protein" covers both small and large polypeptides (210 Kb). Obviously, it is more likely for large polypeptides that they contain both reactive amino and carboxyl groups without any modification.

According to the essence of the invention, the term "native polysaccharide" refers to a saccharide that has not been subjected to processes aimed at reducing the size of the saccharide. The polysaccharide may become slightly smaller in size during normal purification procedures. Such a saccharide remains native. Only if the polysaccharide has been subjected to fractionation processes, then such polysaccharide will not be considered as native.

According to the essence of the invention, the expression "fractionation with a factor of up to x 2" means that the saccharide is subjected to the predicted process of reducing the size of the saccharide but maintains a size of more than half the size of the native polysaccharide. x3, x4, etc. interpreted in the same way, that is, the saccharide undergoes the predicted process of reducing the size of the saccharide but maintains the size by more than a third, a quarter, etc. size of the native polysaccharide.

The period of time from 35 seconds to 6 hours in step 5) of the method for adding a complete aliquot of the final component can be from 50 seconds to 5 hours, from 1 minute to 4 hours, from 2 minutes to 3 hours, from 3 minutes to 2 hours, from 4 to 60 minutes, 5 to 50 minutes, 6 to 40 minutes, 7 to 30 minutes or 8 to 20 minutes. It can also be 1 minute to 5 hours, 10 minutes to 4 hours, 20 minutes to 3 hours, 30 minutes to 2 hours, 40 to 90 minutes, or 50 to 70 minutes. This time can be adjusted according to the specific saccharide and protein introduced into the condensation.

In one embodiment, an aliquot of the final component (eg, carbodiimide, saccharide, or protein) is added to the reaction mixture at a constant rate over a period of time (easily accomplished using a constant rate pump). Alternatively, it can be added in several portions over a period of time. Although this can be done in many ways, in the main aliquots should be added throughout the period. For example, at least one quarter of the aliquot may be added during the first half of the period, and at least one quarter of the aliquot during the second half of the period. The total amount of measured aliquot a", for example, in ml or mg can be added in 4-100 stages (5) over a period, in one of the embodiments, the stages are systematized so that equal amounts (a/5) are introduced at all stages. In one of the embodiments stages are evenly spaced with intervals over the period "p" (in seconds).

Thus, if one stage occurs at time zero with a period of "p", then each subsequent stage could occur at a time equal to p/(5-1). The volume of an aliquot of the final component added in stage B) can be adjusted by the convenience of addition aliquots to the reaction with the desired time period. The carbodiimide can be added as an aqueous solution (usually buffered at pH 7.5 before addition to the reaction) or as a solid powder (EOAS, for example, is highly soluble in aqueous media). Of course, if the carbodiimide is the last component, added to the reaction (option I stage B)), slow dissolution of the carbodiimide can be used so that a full aliquot of the powder is added to the reaction all at once, but it dissolves at a rate consistent with the desired period over which the aliquot becomes available for the reaction.

If the protein and/or saccharide does not contain an amino or carboxyl group (or only one of them), it can be modified to introduce one into it (or to introduce another if it did not contain one). For example, only for a saccharide containing reactive hydroxyl groups (eg meningococcal serogroup A capsular saccharide) would such a group be used to modify amino or carboxyl groups, so much so that EOAS condensation can occur.

This may occur in repeating subunits or may be a group present only at the end of the saccharide molecule.

As would be noted, where modification occurs, only the partially modified half can be useful. For saccharides with repeating subunits, the target epitope may be present in each repeat. Therefore, if partial modification occurs (by this means 0.5-20, 1-15, 3-12, or 5-1095 of the reactive group of the target is actually modified), then this may have the advantage of retaining most of the epitopes and significantly preventing cross-linking .

If a saccharide or protein already has only an amino or carboxyl group (eg Mi saccharide from ZaItopea urni which naturally contains carboxyl but not amino groups), modification can occur to give it a different type of group (ie amino group for Mi). It was noted, however, that as a modification may be partial this action may change the prevailing reaction of the invention from the type | on type I. For example, if Mi saccharide is conjugated to a carrier protein that includes both amino and carboxyl groups, the variant | slowly adds an aliquot of protein in stage b). If the carboxyl group of Mi saccharide is partially modified with amino groups, then it will contain both carboxyl and amino groups, thus option I, slow addition of an aliquot of carbodiimide in step p) becomes the most important.

Modification can occur through the addition of a hetero- or homo-bifunctional linker. This may follow similar chemical processes as described above for the saccharide-protein conjugation step (for example, by the carbodiimide or SOAP method). The linker may contain 4 to 20, 4 to 12, or 5 to 10 carbon atoms, it may contain two reactive amino groups, two reactive carboxyl groups, or one of each (eg, hexanediamine, b-aminocaproic acid, or adipic acid hydrazide ). Typically, modification occurs by reacting a large excess of linker with a saccharide and/or carrier protein to be modified. This allows the modification to proceed with minimal internal cross-linking (which may be possible if, for example, a carboxyl group on a saccharide is modified with amino groups using a carbodiimide condensation). Excess linker is easily removed using a technique such as diafiltration.

In one embodiment, the saccharide includes a reactive hydroxyl group as part of its repeating moiety, which is partially modified through an amino group on the linker (eg, by the SOAP method). In another embodiment, the saccharide includes a reactive amino group as part of its repeating moiety that is partially modified through a carboxyl group on the linker (eg, by the carbodiimide method). In further embodiments, the saccharide includes a reactive carboxyl group as part of its repeating moiety that is partially modified through an amino group on the linker (eg, by the carbodiimide method).

The aliquot of carbodiimide to perform the conjugation (present in either step a) or B) of the reaction of the invention) was 0.01 to 3,4).05 to 2, or 0.09 to 1 mg carbodiimide/mg saccharide.

Although these amounts were calculated relative to EBAS as a carbodiimide, these amounts can be adjusted if any other carbodiimide is used by multiplying the amount in the series: (molecular weight of other carbodiimide)/X molecular weight of EOAS).

In general, the saccharide can be present in the methods of the invention with a final concentration of 0.5-50 mg/ml in stage p). This will depend on the size and nature of the saccharide, and the degree of any modification. For example, a high concentration will be necessary for oligosaccharides, but a much lower concentration will be more acceptable for large polysaccharides. If it has a large end partially modified with amino or carboxyl groups, then a lower concentration may be acceptable to reduce the possibility of any cross-linking. The carrier protein may be present with a final concentration of 1-50 mg/ml in stage B).

The initial ratio of carrier protein to saccharide in the methods of the invention may be 5:1 to 1:5, 41 to 1:11, or 3:1 to 2:1 (m/m). Furthermore, it will depend on the size and nature of the saccharide and the degree of any modification.

Salt conditions (eg mass) may also vary depending on the nature of the saccharide/protein.

Usually, about 0.2 M of Mass can be found in stage 5) of the methods according to the invention, but it can be 0-2, 0.1-1 or 0.2-0.5 M.

Within the range of pH in stage B) of the methods according to the invention, the pH of the reaction can be any pH where carbodiimide is activated - for example, pH 4.5-6.5, 4.7-6.0, or 5-5.5. This pH is usually maintained throughout the reaction by adding acid/base as needed. EOAS is generally stable at pH 7.5, although if conjugation is required at higher pH, compounds known to keep the reaction medium stable (such as M-hydroxysuccinimide) may also be present in stage B), in these cases the pH of the reaction in stage b) can be kept at pH 4.5-7.5.

The reaction temperature during stage B) of the methods according to the invention can be 4-37, 10-32, 17-30, or 22-27"C, and as a rule, it is maintained during the reaction.

In the methods of the invention, a full aliquot is added once in step B), the reaction is generally maintained for a further 10 minutes to 72 hours, 20 minutes to 48 hours, 30 minutes to 24 hours, 40 minutes to 12 hours, 50 minutes to 6 hours , or 1-3 hours. Sometimes the reaction is complete, the pH is adjusted to 7.5-9 (if M-hydroxysuccinimide is present, then to its higher end) to return to the pH range in which the carbodiimide is stable.

Sometimes the conjugated saccharide-protein conjugate can be purified from: unreacted components, free saccharide, etc. by injecting it into a 5i2e exsiysiop spgotaidharpusoishtp (for example, Cefacryl

З40ООНЕ, РПагтасия)" As a rule, this is performed at 2-8 "С. The conjugate is sterile filtered, then stored. Finally, an effective dose (for example, 1-20, 2-15, or 3-10 μg of saccharide / dose) A saccharide-proin conjugate can be formulated with a pharmaceutically acceptable excipient (eg, salt or excipient) to produce an immunogenic composition or vaccine.

In terms of saccharides of this invention, any saccharide of viral, tubular, bacterial or eukaryotic origin can be conjugated using the methods of the invention. It may be the Mi saccharide from Zaitopeia gurni, or a saccharide other than Mi. It can be the capsular saccharide NIB from N. ipisepagae type p, or it can be a saccharide other than NIB. In one embodiment, the saccharide is a bacterial capsular saccharide, for example, isolated from a bacterium selected from the list consisting of: M. tepipoiaiv zegodgoir A (MepA), B (Mepv), C (Meps), UM135 (Mepum) or y ( Mep), Bigeriosossi5 rpeitopiae serotypes 1, 2, 3, 4, 5, bA, 68, 7E, 8,

OM, 9M, 10A, 11 A, 12E, 14, 158, 17, 18C, 19A, 19E, 20, 22E, 23E or ZZE, Group B Streptococcal group ia, IB, II,

I, IM, M, MI, or MII, Eharpuisossiv acigeiv type 5, Ziarpuisossiv acigeiv5 type 8, ZaItopeia iurpi (Mi saccharide),

Mirpo spoiegaye, or N. ipYaiicepaye type br.

The weight average molecular weight of the saccharide can be 1000-2000000, 5000-1000000, 10000-5O0000, 50000-400000, 75000-300000, or 100000-200000. The molecular weight or average molecular weight of the saccharide is here assigned to the weight average molecular weight (Mm/) of the saccharide measured before conjugation and which is measured by the MAGI 5 method. The MAGI/5 method is well known in the art and is generally performed as described in Example 2 For MAG 5 analysis of saccharides, two columns can be used in combination (T5KO6000 and 5000RMUKHI) and elute saccharides with water. Saccharides are detected using a light scattering detector (e.g., Uuwai Oamp O5R equipped with a 10mW argon laser with a wavelength of 488nm) and an iptegotheis refractometer (e.g., Uuwai O(Shar O5R equipped with a PI100 cell and a red filter at a wavelength of 498nm). In an embodiment, polydispersity saccharide is 1-1.5, 1-1.3, 1-1.2, 1-1.1 or 1-1.05 and after conjugation to the protein carrier, the polydispersity of the conjugate is 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.0-1.2 , 1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements were made by the MAG 5 method.

The saccharide can be either a native polysaccharide or can be fractionated by a factor of no more than 2, 4, 6, 8, 10, or 20 times (for example, by microfluidization (for example, on the Ety5Shekh C-501 device or by other known methods) and, for example, heating methods, (chemical, oxidizing, sonication!) Oligosaccharides, to a large extent, can be fractionated into fractions (for example, known methods of heating, chemical or oxidizing)|.

The structures of most of these saccharides are known (and therefore either naturally contain any amino or carboxyl groups for the carbodiimide technique, or any other reactive group that can be modified with amino or carboxyl groups (see table below).

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The saccharide can be a bacterial lipooligosaccharide or lipopolysaccharide (see the table above), for example, isolated from a bacterium selected from the list consisting of: M. tepipouliai5, N. ipJaiceplae, E. soi,

ZaItopeia or M. saiagtaiv. GO5 can be meningococcal immunotype 1/2, 3 or 10. It can be detoxified by alkaline treatment of its Lipid A part.

In an embodiment, the MepA capsular saccharide is at least partially O-acetylated such that at least 5095, 6095, 7095, 8095, 9095, 9595 or 9895 repeating fragments are O-acetylated at at least one position. O-acetylation, for example, is present at least in the 0-3 position of at least 5095, 6090, 7090, 8095, 9095, 9595, or 98905 repeating fragments. In an embodiment, the Meps capsular saccharide is at least partially O-acetylated such that at least 3090, 40905, 50905, 6090, 7090, 8090, 90905,

95905 or 9895 of the (c2--9)-linked MenMmAs repeating fragments are O-acetylated at least in one or two positions. O-acetylation is, for example, present at the 0-7 and/or 0-8 position of at least 30905. 40905, 50905, 6090, 7095, 8095, 90906, 9595 or 9895 of the repeating fragment. In an embodiment, the Mepu/capsular saccharide is at least partially O-acetylated such that at least 3095, 4095, 5095. 60905, 7090, 8095, 9095, 9590, or 9895 of the repeating fragments are O-acetylated. in one or two positions. O-acetylation, for example, is present at positions 0-7 and/or 0-9 of at least 3095. 40905, 50905, bout, 7095, 8095, 9095, 9590, or 9895 repeating fragments. In an embodiment, the Mepu capsular saccharide is at least partially O-acetylated such that at least 2095, 30905, 4095, 5095, 6095, 7090, 8090, 9095, 9595, or 9895 repeating fragments are O-acetylated. in one or two positions. O-acetylation is present at the 7 and/or 9 position in at least 2095, 3090, 4095, 5090, 6090, 70905, 8095, 9090, 9590, or 9895 repeat fragments. The percentage of O-acetylation refers to the percentage of repeating fragments that contain O-acetylation. This can be measured in the saccharide before conjugation and/or after conjugation.

The carrier protein can be any peptide or protein. It may contain one or more T-helper epitopes. In one of the embodiments of the invention, the carrier protein is selected from the group consisting of: TT, Ot,

SAM197, fragment C from TT, protein O from N. ipishepgaye, pneumococcal RIPOY, and pneumococcal Rpeshtoiuviv.

The protein carrier can be tetanus toxoid (TT), fragment C of tetanus toxoid, non-toxic mutants of tetanus toxoid (it is believed that all such variants of TT are the same type of protein carrier in the essence of the present invention|, diphtheria toxoid (07), SCM197, other non-toxic mutants of diphtheria toxoid (such as SEM176, SKM 197, SKM228, SEM 45 (spida ei a! 9. Vioi. Spet. 218; 3838-3844. 1973);

SAM 9, SKM 45, SEM102, SEM 103, and SEM107 and other mutants described by Mispoi5 and Mocie in sepeisaiu

Epdipeegei Tohip5, Ed: Egapkei, Maesei! Oeckeg Ips. 1992; deletion or mutation of SiIc-148 to Avr, bip to 5eg and/or

Aa 158 to Su and other mutations revealed in 5 4709017 or 5 4950740; mutations of at least one or more residues of Guz 516, Guz 526, Rpe 530 and/or I uz 534 and other mutations disclosed in 5 5917017 or 05 6455673; or the fragment disclosed in 05 5843711) (it is believed that all such variants of OT are the same type of protein source in the essence of the present invention), pneumococcal pneumolysin (Kyo eyi a! (1995) Iptesi Ittip 63; 2706-13), OMRSO (meningococcal surface membrane protein - usually isolated from M. tepipouiids zegodgotspr B -

EROZ372501), synthetic peptides (EROZ378881, ERO427347), heat shock proteins (MO 93/17712, MO 94/03208), pertussis proteins (MO 98/58668, ERO471177), cytokines, lymphokines, growth factors, hormones (MO 91/01146) , unnatural proteins that include multiple human T cell epitopes from various pathogenic derived antigens (Raitschdi ei a! (2001) Yeig U Ittipoi 31; 3816-3824) such as M19 protein (Vagaidoi ei a! (2004) Iptesi Ittip 72 ; 4884-7) pneumococcal surface protein RerA (UMO 02/091998), iron-injecting proteins (MO 01/72337), toxins A or B with C UMO 00/56360), pneumococcal RITA (M/O 98/18930, also submitted for consideration to 5rz3b) pneumococcal

PtTO (disclosed in UMO 00/37105, and also submitted for consideration to zroz360), pneumococcal RYIV (disclosed in

UMO 00/37105, and also submitted for consideration to 5roz6c), or RICHE (disclosed in ММО00/30299 and submitted for consideration to as VMN-3).

In further approaches of the invention, a saccharide-protein carrier conjugate (or immunogenic composition or vaccine) is provided, which can be obtained, or obtained by the methods of the invention.

The use of the immunogenic composition or vaccine of the invention in the production of a medicine for the prevention or treatment of a disease, and the method of prevention or treatment of a disease, including the stage of regulating the effective dose of the immunogenic composition or vaccine of the invention for the patient, is further provided if necessary. The use or method may be such that the disease is the case of a selected bacterium from the list consisting of: M. tepipouyaiv, Zigeriosossiv rpeshtopiae, M. saiagptpaiyev. Group B Zgeriosossyv,

Zarpuiosossiv atsygeiv. ZaItopeia iurpi, Mirgpo spoiegae, E. soi, and N. ipiseplgae.

The immunogenic compositions of the invention may also contain an OTR or OTRm/ vaccine (for example, one including OT, TT, and either a whole cell pertussis (Ru) vaccine or a non-acellular pertussis (Ra) vaccine (including, for example, pertussis toxoid, ENA, pertactin , and, optionally, agglutinogens 2 and 3). Such combinations may also include hepatitis B vaccines (for example, they may contain hepatitis B surface antigen) (Nervi, optionally sorbed on aluminum phosphate). includes Nir, MepA and Meps saccharide conjugates, or Nir and Meps saccharide conjugates, or

NiB, MepC and Mepu saccharide conjugates, or MepA, MepC, MepuUu/ and Mep saccharide conjugates, in which at least one, two or all saccharide conjugates are obtained according to the method of the invention.

The immunogenic compositions of the invention optionally include additional viral antigens that provide protection against or against disease caused by measles and/or mumps and/or rubella and/or chicken pox.

For example, the immunogenic composition of the invention contains antigens of measles, mumps and rubella (MMK) or measles, mumps, rubella and chickenpox (MMEM). In an embodiment, these viral antigens are not necessarily present in the same content as present in the meningococcal and/or NIb saccharide conjugate(s) composition. In one embodiment, these viral antigens are lyophilized.

In one of the embodiments, the immunogenic composition of the invention further includes an antigen from M. tepidiai serogroup

B. The antigen is optionally from the outer membrane of the vesicle prepared from M. tepidinae serogroup B, as described in ERZ301992, UMO 01/09350, UMO 04/14417, MO 04/14418 and UMO 04/14419. basically, the immunogenic composition of the invention may include a dose of each saccharide conjugate between 0.1 and 20 μg, 2 and 10 μg, 2 and 6 μg, or 4 and 7 μg of saccharide. "Near" or "near" is determined from 1095 more or less than this figure according to the purpose of the invention.

In one embodiment, the immunogenic composition of the invention is maintained either by a buffer at or maintained between pH 7.0 and 8.0, pH 7.2 and 7.6, or near or exactly pH 7.4.

The immunogenic composition or vaccine of the invention is optionally lyophilized in the presence of a stabilizing agent, for example, a polyol such as sucrose or trehalose.

Optionally, the immunogenic composition or vaccine according to the invention contains an amount of adjuvant sufficient,

to enhance the immune response to the immunogen. A suitable adjuvant includes, but is not limited to, aluminum salts (aluminum phosphate or aluminum hydroxide), squalene mixture (ZAE-1), muramipeptide, saponin derivatives, mycobacterial cell wall preparations, monophospholipid A, mycolic acid derivatives, nonionic block copolymer surfactants - active substances (surfactants), Options A, B subunit of cholera toxin, polyphosphazene and its derivatives, and immunostimulating complexes (IZSOM5), such as those described in Takapavni ei ai. (1990) Maishge 344:873-875.

For M. tepipoyaiv or NirMep combinations, it may be acceptable to use no aluminum salt as adjuvant or no adjuvant at all.

As with all immunogenic compositions or vaccines, immunologically effective amounts of immunogen must be determined empirically. Factors, as noted, include immunogenicity, optionally the immunogen will be complexed with or covalently attached to an adjuvant or a carrier protein or other carrier, the method of administration and the number of immunizing doses administered.

The active agent may be present in various concentrations in the pharmaceutical composition or vaccine of the invention. Generally, the minimum concentration of a substance is the amount required to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or be homogeneously suspended with the original mixture. For example, the minimum amount of a pharmaceutical agent is not necessarily one that will provide a single therapeutically effective dose. For bioactive substances, the minimum concentration is the amount required to detect bioactivity, and the maximum concentration is at the point at which the suspension cannot be kept homogeneous. In the case of single-dose units, the amount is the same as for a single therapeutic use. In general, it is expected that each dose will include 1-100 mcg of protein antigen, not necessarily 5-50 mcg or 5-25 mcg. For example, doses of bacterial saccharides are 10-20 μg, 5-10 μg, 2.5-5 μg or 1-2.5 μg of saccharide in the conjugate.

Vaccine preparations according to the present invention can be used to protect and treat mammals (for example, humans) susceptible to infections, by the method of introducing said vaccine systemically or through the mucous membrane. Human patients - not necessarily infants (up to 12 months), toddlers (12-24, 12-16, or 12-14 months), children (2-10, 3-8, or 3-5 years), young adults (12-21, 14-20 or 15-19 years old) or adults. These administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous administration; or by introduction through the mucous membrane - oral/alimentary, respiratory, genitourinary tracts.

Intranasal administration of the vaccine for the treatment of pneumonia or otitis media is preferred (as the nasopharyngeal carrier of pneumococci can be more effectively prevented, thus weakening the infection at the earliest stage). Although the vaccine of the invention may be administered as a single dose, the components thereof may also be administered together at the same time or at different times (eg, if saccharides are present in the vaccine, they may be administered separately at the same time or 1-2 weeks after administration of the bacterial protein vaccine for optimal coordination of the immune response with one-to-one correspondence). In addition to the single route of administration, 2 different routes of administration can be used. For example, viral antigens can be administered IO (intradermally), while bacterial proteins can be administered IM (intramuscularly) or IM (intranasally). If saccharides are present, they can be administered IM (or I) and bacterial proteins can be administered IM (or Id). In addition, the vaccines of the invention can be administered IM for priming doses and IM for booster doses.

The preparation of the vaccine is mainly described in Massipe Oezidp ("Tpe z!yBb-ipii apa adiimapi arrgoasn" (eav

Romans! M.R. " Memltap M.9.) (1995) Riepyt Rgez5 Mem HogK). Encapsulation with liposomes is described by Eshepop, O5

Raiepi 4,235,877.

A further aspect of the invention is a process for manufacturing an immunogenic composition or vaccine according to the invention, which includes the step of mixing MepA and Meps saccharides according to the invention, produced by the method according to the invention, with Mepuu and Mepu not produced according to the invention, and with a pharmaceutically acceptable excipient.

By the terms "comprising", "comprising" and "comprising" the applicant means the optional substitution of the terms "consisting of", "consisting of" and "consisting of", respectively, in each example.

All references or patent applications cited in this description of the invention are incorporated by reference herein.

The invention is illustrated by the attached examples. The examples below were performed using standard techniques that are well known and familiar to those skilled in the art, with exceptions described in detail.

The examples illustrate but do not limit the invention.

Examples

Example 1 - preparation of polysaccharide conjugates

Example Ta - preparation of meningococcal MepA and MepC capsular polysaccharide conjugate according to the invention

Meps-TT conjugates were produced using native polysaccharides (over 150 KOa as measured by

MAG 5) or slightly microfluidized. MepA-TT conjugates were produced using either the native polysaccharide or a slightly microfluidized polysaccharide over bOkOa as measured by the MAG 5 method of Example 2. Fractionation was performed by microfluidization using an Eta!5Sheh C-50 homogenizer. The polysaccharides were then filtered through a 0.2 µm filter .

In order to conjugate MepA capsular polysaccharide to tetanus toxoid through a spicer, the following method was used. Covalent binding of polysaccharide and spicer (AOH) was carried out by chemical coupling, according to which polysaccharides are activated under controlled conditions by a cyanating agent, 1-cyano-4-dimethylamino-pyridine tetrafluoroborate (SOAR). Spicer reacts with cyanated PO through its hydrazo groups, forming a stable isourea bond between spicer and polysaccharide. 10 mg/ml MepA solution (pH 6.0) and 3.5 g was treated with a freshly prepared 100 mg/ml solution of SOAR in a mixture of acetonitrile/water (50/50 (0/0)) to obtain a ratio of SOAR/MepaA of 0.75 (m/m). After 1.5 minutes, the pH was raised to 10.0. Three minutes later, AOH was added to obtain a ratio of AUN/MepA of 8.9... the pH of the solution was lowered to 8.75 and the reaction continued for 2 hours maintaining this pH (with the temperature maintained at 25 70).

The RBZAAdn solution was concentrated to a quarter of the initial volume and then diafiltered with 30 volumes of a 0.2M mass solution using a Riigop-Oteda membrane with pores of 10 Kba, and the retentate was filtered.

Before the conjugation reaction (carboimide condensation), the purified TT solution and ROZAdn solution were diluted to reach a concentration of 10 mg/ml for RbBAdn and 10 mg/ml for TT. EOAS (i-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added to the RBAN solution (2 g of saccharide) in order to achieve a final ratio of 0.9 mg EOAS/mg RoAdn. The pH was adjusted to 5.0. Purified tetanus toxoid was added using a peristatic pump (after 60 minutes) to achieve 2 mg TT/d RbAdn. The resulting solution was left for 60 min at 25"C with shaking so that the final total time was 120 min. The solution was neutralized by adding a 1M solution of Ttgiz-Nei pH 7.5 (1/10 of the final volume) and left for 30 minutes at k25"C, and then overnight at a temperature from t27C to 80.

The conjugate was made transparent using a 10 μm pore filter and purified on a Sephacryl 540ONE column (RPpagtasia, Zul"edep). The column was equilibrated in 10 mM Ttg5-HCl (pH 7.0), 0.075 M mass solutions and the conjugate (about bbOml ) were loaded into a column (from 42°C to 88°C). Pelletization was selected based on the value of the optical density at 280 nm. Collection began when the absorbance increased to 0.05. Collection was continued until Ka 0.30 was reached. then stored from 12"C to 8"C. The resulting conjugate had a polysaccharide:protein ratio of 1:2-1:4 (m/m).

In order to conjugate Meps capsular polysaccharide to tetanus toxoid through a spicer, the following method was used. Covalent binding of polysaccharide and spicer (AOH) is performed by chemical coupling, by which the polysaccharide is activated under controlled conditions by a cyanulating agent, 1-cyano-4-dimethylamino-pyridine tetrafluoroborate (SOAR). The spicer reacts with cyanated Pb5 through its hydrazo groups, forming a stable isourea bond between the spicer and the polysaccharide. A 20mg/ml solution of Meps (rnb.0) (3.5 g) was treated with a freshly prepared 100mg/ml solution of SOAP in a mixture of acetonitrile/water (50/50 (0/0)) to obtain a ratio of SOAP/MepsC of 1.5 (m/m). After 1.5 minutes, the pH was raised to 10.0. At pH activation, 5M Masi solution was added to reach a final concentration of 2M Masi solution. Three minutes later, AOH was added to give an AOH/Meps ratio of 8.9. The pH of the solution was lowered to 8.75 and the reaction continued for another 2 hours (kept at 25°C).

The RZSdn solution was concentrated to at least 150 ml, and then dialyzed with 30 volumes of a 0.2M mass solution using a Riigop Oteda membrane with 10 Kkba pores, the retentate was filtered.

Before the coupling reaction, the purified TT solution and ReZSadn solution (2g scale) were diluted in a 0.2M solution

Mass until reaching a concentration of 15 mg/ml for ROSdn and 20 mg/ml for TT.

Purified tetanus toxoid was added to the RZSadn solution in order to reach 2 mg TT/mg RoSadn. pH is brought up to 5.0. EOAS (16.7 mg/ml in Tgi5 0.1 M pH 7.5) was added using a peristaltic pump (after 10 minutes) to achieve a final ratio of 0.5 mg EOAS/mg ROS. The resulting solution was left for 110 min at 25°C with shaking and pH adjustment to obtain a final total time of 120 min. Then the solution was neutralized by adding a 1M solution of Ttgi5-Hsi pH 9.0 (1/10 of the final volume) and left for 30 minutes at 25°C, and then overnight from 2°C to 8°C. The conjugate was made transparent using a filter with pores of 1 Ωm, and purified on a Sephakryl 5400NK column (RPpagtasia, Zmedep). The column was equilibrated in a 10 mM Tgiz-HCII solution (pH 7.0), 0.075 M Mass, and the conjugate (about 460 ml) was loaded into the column (from 127°C to 8°C). The elution pool was selected based on the value of the optical density at 280 nm. Collections began when the absorbance increased to 0.05. Collection was continued until reaching Ka 0.20. The conjugate was filter sterilized at 20°C, then stored from 12°C to 18°C. The resulting conjugate had a polysaccharide-protein ratio of 1:2-1:4 (m/m).

A variety of experiments were performed with the addition of ESAC for more than 10-45 minutes - in each case, good quality MepsS conjugates were obtained. If, however, the TT carrier was slowly added last to the Meps-

ARON 5 EOAS, this led to a gel - conjugate that could not be purified.

Experiments were also performed by adding ECAS all at once to the reaction, but the final T/P5 ratio (2.7/1) (m/m) of the conjugate was lower than that of the conjugate obtained through the reaction where ECAS was added over 10 minutes (3.3/1 ); in addition, both "TT" and "xP5" were lower than those measured for the conjugate obtained by the reaction where ESAC was added for more than 10 minutes.

State the approximate 96 Modifications of polysaccharides

MepsAN: after SOAR treatment with AOH, about 3.4795 hydroxyl groups were modified with AOH (based on two available hydroxyl groups per repeating subunit). For MepA: about 11,595 hydroxyl groups modified with AOH (taking into account the presence of only one available hydroxyl group per repeating unit).

Example 16 - preparation of pneumococcal capsular RB5 From polysaccharide conjugate 1) RBOZ-TTan process: RBOZ-TTaln2gOVv

Fractionation on EEtyi5ziyeh

RBZ was weighed based on 1095 theoretical moisture content. Native P5 was dissolved overnight in 2M Massey solution to an initial concentration of 3 mg/ml. Before fractionation, the solution of native PO was purified on a filter with pores of 5 μm.

Homogenizer EMYOG5BIRSEH 0-50 was used to reduce the molecular weight and viscosity of the polysaccharide before the activation stage. The fractionation efficiency depends on the circulating pressure, the feed plunger pressure and the total number of cycles. In order to improve the efficiency of the fractionation (and therefore to reduce the total number of cycles), the homogenizing cells of Etii5ziyeh were replaced by cells of fixed geometry (Misgoichdiyev E20u-0.75 μm interaction chamber). The purpose of fractionation is to reduce the molecular weight and viscosity of P5 without critically reducing its antigenicity.

The size of the contraction was done at 6000 x 500 psi (pounds per square inch) and subsequent in-process viscosity measurements. Fractionation was stopped when 2.0 20.2 er was reached.

Filtration of fractionated RBZ by 0.22 μm

Fractionated P5 was filtered on a MiPirask 40 membrane (pore size 0.22 mm) at a flow rate of 10 ml/min.

TT modification

The modification stage was carried out at 257C with constant shaking in a water bath with temperature control. TT was diluted in 0.2M Masi solution to obtain a final TT concentration of 25 mg/ml. ABSN was added in solid form to the TT solution to reach a final concentration of 0.2M After complete dissolution

AVN, the pH of the solution was set to 6.2-/- 0.1, using a solution of NSI.

EOVAS was then added to the TT/AOH solution to reach a final concentration of 0.02M. The pH was set to 6b.2---0.1 using a solution of NCI and maintained by adjusting the pH for 1 hour.

After the modification step, the pH was raised to pH 5 using Mahon's solution to stop the reaction.

The solution was left for 2 hours with pH adjustment before the diafiltration stage.

Diafiltration

The TTdan derivative was diafiltered to remove unreacted AOH and EOAS coproducts.

Diafiltration was performed on a SEMTVAMATE membrane (0.09 m, with pores of 10 kba). The solution was dialyzed against the background of volumes of 0.2M mass solution.

The next stage of diafiltration was performed by quantitative analysis of AOSN (TMB5 analysis) in penetration after 5, 10, 15 and 20 volumes of diafiltration.

0.22 micron filtration

Finally, TTan was filtered on a membrane (MiPirask 40) with pores of 0.22 at a flow rate of 10 ml/min.

Then the filtered TTan was stored at -707C.

RBZZ-TTadn conjugate

The conditions of the process were as follows:

Initial concentration of P5ZZ 2 mg/ml in 2 M Massi solution, initial TTAn/RZZ ratio 1.5/1 (m/m),

EBAS concentration of 0.5 mg/mg RB, and TT concentration of 10 mg/ml in a 0.15 M solution of mass. 50 mg of RBZZ was diluted in a 2 M mass solution to obtain a final concentration of P5 of 2 mg/ml. The purified TTadn solution was diluted in 0.2M mass solution to reach a concentration of 10 mg/ml. The pH of the P5ZZ solution was adjusted to 5 using a solution of NCI.

EBAS was added in a solid state to the P5Z solution in order to reach a final concentration of 0.5 mg EBAS/mg RB. The pH was adjusted to 5.0 x 0.05 using a solution of NCI and TTAN was added manually after 11 minutes (aliquot/min).

The resulting solution was incubated for 109 min at 25°C with shaking and pH adjustment to obtain a final total time of 120 min. Then the solution was neutralized by adding a 1M solution of Ttgiz-HCl pH 7.5 and left for 30 min at 25°C. The conjugate was finally purified (made transparent) on a membrane with pores of 5 μm and injected into a Sephakril 540OONE column. 2) RBOZ-TTap process: RZOZAN-TT215

Fractionation on EEtyi5ziyeh

As stated above.

Filtration of fractionated RBZ by 0.22 μm

As stated above. ROZ modification

The modification stage was carried out at 257C with constant shaking in a water bath with temperature control T"

ROZ was set to 6.0 before adding SOAP (0.25 mg/mg P5, dissolved to 100 mg/ml in a mixture of acetonitrile/LughI). The pH was raised to 9.5 using Mahon before the addition of AOH (8.9 mg AOH/mg RB, dissolved to 100 mg/ml in 0.2M Massey's solution). The pH was kept at 9.5 and adjusted for 60 minutes.

The percentage of modification corresponded to 2.495 (2.4 mg AOH/100 mg P5). This can be measured according to the known method:

ТМВ5 for assessment of AON; and OMAV or resorcinol (Mopzidpu eyi a! (1988) Apa!. Viospet. 175, 525-530) for quantitative analysis of P5. In this case, TMB5 dosage was 228 μg/ml and P5 dosage: 5250 μg/ml.

The submitted My AON is 174.2, and the Mu of the repeating fragment P5ZZ is 338.27 (having 1

COOH and 4 OH groups), this is 1.3 μmol of AOH / 15.52 μmol of repeating fragment, or 1.3 μmol of AOH / 62.08 μmol of reactive hydroxyl group. 2.0995 РОЗ of hydroxyl groups were AOH modified hydroxyl groups.

Diafiltration

ROZdn derivatives were diafiltered in order to remove unreacted AOH and SOAR coproducts.

Diafiltration was carried out on a membrane SHER-30-S-H2AG A (42 cm", with pores of 30 kba). The solution was dialyzed against the background of 20 volumes of 0.2 M Massey's solution.

The next stage of diafiltration was performed by quantitative analysis of AOH (TMB5 analysis) in the penetration after 5, 10, 15 and 20 volumes of diafiltration.

0.22 micron filtration

Rbdn was finally filtered on a membrane (MiPirask 40) with pores of 0.22 μm at a flow rate of 10 ml/min.

Then the filtered RZZadn was stored at 4-7s.

RZZadn-TT conjugate

The conditions of the process were as follows:

The initial concentration of P5ZZ is 2 mg/ml in a 2 M solution of Masi, the initial TT/RZZadn ratio is 1.5/1 (m/m),

EBAS concentration of 0.5 mg/mg RB, and TT concentration of 10 mg/ml in 0.15 M mass solution. 5mg of RZZdn was diluted in a 0.2M mass solution to obtain a final concentration of RB5 of 2 mg/ml. Peeled

The TT solution was diluted in 0.2M Massey solution to reach a concentration of 10 mg/ml. The diluted solution was adjusted to pH 5 using a solution of NSI. EVAS was also added in the solid state to the RoZdn solution in order to reach a final concentration of 0.5 mg EOAS/mg P5. The pH was adjusted to 5.0 x 0.05 with NCI solution and TT was added after 10 minutes using a peristaltic pump. The resulting solution was incubated for 110 min at 2570 with shaking and pH adjustment to give a final total time of 120 min. Then the solution was neutralized by adding a 1M solution of Ttgiz-HCI, pH 7.5 and left for 30 min at 2570. The conjugate was finally purified (made transparent) on a membrane with pores of 5 μm and injected into a Sephacryl 540ONE column.

C) RBOZAN-TT rgosev5: ROZAdn-TT217

Fractionation on EEtyi5ziyeh

As stated above.

Filtration of fractionated RB on 0.22 μm

As stated above. RZZ modification

As for the 215 conjugate.

Diafiltration

As for the 215 conjugate.

0.22 micron filtration

As for the 215 conjugate.

RZZadn-TT conjugate

The conditions of the process were as follows:

The initial concentration of P5ZZ is 2 mg/ml in a 2 M solution of Masi, the initial TT/RZZadn ratio is 1.5/1 (m/m),

EBAS concentration of 0.5 mg/mg RB, and TT concentration of 10 mg/ml in 0.15 M mass solution. 5mg of RZZdn was diluted in a 0.2M mass solution to obtain a final concentration of RB5 of 2 mg/ml. Peeled

The TT solution was diluted in a 0.2M mass solution to reach a concentration of 10 mg/ml. RZZdn and TT solutions were mixed and the pH was adjusted to 5 using a solution of NSI. EOAS was dissolved in Tgiz 1M buffer pH 7.5. 40 μl of ЕВСАС was added every minute (10 minutes to reach the ЕВАS/RB ratio (0.5 mg ЕОАС/mg RB)). The resulting solution was incubated for 110 min at 25"C with shaking and pH adjustment to obtain a final total time of 120 min. Then the solution was neutralized by adding a 1M solution of Tgi5-HCl pH 7.5 and left for 30 min at 2570. Kon' the yugat was finally purified (made transparent) on a membrane with pores of 5 μm and injected into a Sephakril 5400ONEK column. 4) RBOZAN-TT process: ROZdn-TT218

Fractionation on EEtyi5ziyeh

As stated above.

Filtration of fractionated RBZ by 0.22 μm

As stated above.

RBZZ modification

The modification stage was carried out at 25"C with constant shaking in a water bath with temperature control. ROZ was diluted in a 2M mass solution to obtain a final RO concentration of 3 mg/ml. EVAS was added in the solid state to achieve an EVAS/RB ratio of 0.1 mg/mg P5. After complete dissolution, the pH of the solution was set to 5. Then, after 44 minutes, AON was added (8.9 mg AON/mg RB5, dissolved to 100 mg/ml in a 0.2 M solution by mass), using a peristaltic pump (regardless of the presence of essentially excess

ARrN, direct addition would also be good OK). The pH was maintained at 5.0--/-0.1 and adjusted for 120 minutes (447 - 76). The reaction was stopped by raising the pH to 7.5 using sodium hydroxide. The percentage of modification corresponded to 3.7905 (mg AON/mg RB5). The TMB5 dosage was 220 μg/ml and the P5 dosage was 5902 μg/ml, thus 1.26 μmol AN / 17.44 μmol repeating fragment ("μmol reactive COOH group).

Thus, 7.22956 RZZ of carboxyl groups were AOH modified COOH groups.

Diafiltration

ROZdn derivatives were diafiltered in order to remove unreacted AOH and EOAS coproducts.

Diafiltration was carried out on a membrane SHER-30-C-H2A A (42 cm", with pores of 30 KqOa). The solution was dialyzed against the background of 23 volumes of 0.2M Massey's solution. The next stage of diafiltration was performed by quantitative analysis of AOH (ТМВ5 analysis) in penetrations after 5, 10, 15 and 20 volumes of diafiltration.

0.22 micron filtration

Rbdn was finally filtered on a membrane with pores of 0.22 μm (MiPirask 40) at a flow rate of 10 ml/min.

Then the filtered RZZadn was stored at 4-7s.

RZZadn-TT conjugate

The conditions of the process were as follows:

The initial concentration of RZZadn is 2 mg/ml in a 2 M mass solution, the initial TT/RZZadn ratio is 1.5/1 (m/m),

EBAS concentration of 0.5 mg/mg RB, and TT concentration of 10 mg/ml in a 0.15 M solution of mass. 5 mg of RZZdn was diluted with 0.2 M Masi solution to obtain a final concentration of RB of 2 mg/ml. The purified TT solution was diluted in 0.2M Masi solution to reach a concentration of 10 mg/ml. ROZdn and TT solutions were mixed together. The pH was adjusted to 5.0 x 0.05 using a solution of NSI and EOAS was added manually after 10 minutes (equal aliquots were added every minute). The resulting solution was incubated for 110 min at 2570 with shaking and pH adjustment to obtain a final total time of 120 min. Then the solution was neutralized by adding a 1M solution of Ttgiz-HCI, pH 7.5 and left for 30 min at 2570. The conjugate was finally purified (made transparent) on a membrane with pores of 5 μm and injected into a Sephacryl 540ONE column.

Conclusions:

Various conjugates were obtained using carbodiimide methods at the conjugation stage. The last component added to the reaction solution can be either TT protein or EVAS reagent. The time of addition can affect the resulting conjugates.

RbZdanttT215 8, 217 conjugates:

The same components and conditions were used to prepare both conjugates.

The way in which the last component was added was different. The RoZdnTT217 conjugate resulted in a product that met the ip-migo criteria. It was made by adding EVAS after 10 minutes.

The ROZdanTT215 conjugate, however, could not be filtered on a sterile membrane. For him, the last component added to the reaction medium was TT (after 10 minutes).

The final TT/P5 ratios were very different for both conjugates. (0.98/1 vs. 0.50/1). If ESAC is added first to RoOdn (having both reactive amino and carboxyl groups), then this could lead to internal cross-linking of the hydrazine and carboxyl groups present on the polysaccharide, and thus could result in a more cross-linked conjugate with a weaker the final ratio after adding TT after 10 minutes.

This effect is not observed for RoZanTT217 conjugate. TT unification occurred better when added

EBAS after 10 min, possibly due to less internal cross-linking, and better external cross-linking between the hydrazo groups of RZZadn and the carboxyl groups of the protein.

In the case of conjugate 218, az She ROZ EVAS modification of only partially modified polysaccharide (keeping most of the polysaccharide epitopes intact), against the presence of both reactive amino and carboxyl groups, hence the slow addition of EOAS at the final stage of conjugation is also beneficial.

Slow addition of TT at the final stage of conjugation was beneficial (however) for the 208 conjugate, where the TT was AOH-modified (and includes amino and carboxyl groups) because the ROZ was left with its native reactive -OH and -"СООН groups as part of its subfragment , which is repeated. Addition

EBAS to RBZZ did not have the aforementioned cross-linking effect, but the slow addition of modified

TT resulted in a conjugate with good ip-migo characteristics - see below.

Characteristics of ip-migo: mg/ml ratio of HC.

ZdanOO1 10

Zdnoo| 0.51

ZdnOb2 0.51

E.TT/P5 arbz/ agro agp/arP5

Conjugate ratio Output RB (95) Filter, output (90) Free P5 (bo) (bo) m/m Antigenicity | Antigenicity of 103 wool in 1 051ev 215105 |Y117 11111111127 1111-1111 103 at rev 1611070 216 "relative to 208 conjugate

Example 1c - preparation of 5. miurpi Mi polysaccharide conjugate according to the invention

Fractionation on EEtyi5ziyeh

RBZ was weighed based on 1595 theoretical moisture content. Native P5 was dissolved overnight in

MIRI to an initial concentration of 7 mg/ml. Before fractionation, the solution of native PO was purified on a filter with pores of 10 μm at a flow rate of 50 ml/min. Homogenizer EMIOI5IRSEH 0-50 was used to reduce the molecular weight and viscosity of the polysaccharide before the activation stage. The efficiency of fractionation depends on the circulating pressure, the pressure of the feeding plunger, and the total number of cycles. In order to improve fractionation efficiency (and thus reduce the total number of cycles), the Ethy5Sheh homogenizing cells were replaced with fixed geometry cells (Mysgolicide E20-0.75 μm interaction chamber). The purpose of fractionation is to reduce the molecular weight and viscosity of PO without a critical decrease in its antigenicity.

Shrinkage was measured at 15,000 x 500 psi (psi) and subsequent in-process viscosity measurements. Fractionation is stopped when 5.0 x 0.3 er is reached.

Filtration of fractionated RBZ by 0.22 μm

Fractionated RU is filtered on a Mirask 40 membrane (0.22 mm pores) at a flow rate of 10 ml/min.

Filtered fractionated P5 is stored at -2076.

Modification of polysaccharide mi 1.5 g of fractionated Mi P5 was dissolved at 25 "C in ERI with shaking (5 mg/ml). 13.35 g of AN (8.9 mg

AVN/mM RB5) is added to the Р5 solution. After complete dissolution with a 1M solution of NSI, the pH was adjusted to 5.0 and 0.05.

EBAS (0.1 mg/mg P5) was added in a solid state. The solution was left for 60 minutes at 25"C. Then the solution was neutralized by adding 1M Trigas-HCl pH 7.5, and left for at least 30 minutes at 25"C (maximum 2 hours). It was established that the level of modification is 4.559565 using TMB5 dosage (mg AON/1O0 mg RB5). TMB5 dosage was 200 μg/ml and P5 dosage - 4034 μg/ml; thus 0.0697 μmol of AOH

/16.46 μmol of the repeating fragment (Mm/245). 1.3 μmol AOH / 16.46 μmol reactive

COOH groups per Mi, thus 795 Mi COOH groups were AOH modified COOH groups.

Diafiltration

The ROMidn derivative was diafiltered in order to remove unreacted AN and EVAS coproducts.

Diafiltration was performed on a SEMTRAMATE membrane (0.09 m, pore size 10 kCOa). The solution was dialyzed against 20 volumes of 0.2M Massey solution. The next stage of diafiltration was performed by quantitative analysis of ASN (TMBv5 sample) in penetration after 3.5, 10 and 20 volumes of diafiltration.

0.22 micron filtration

ROMidn at the final stage was filtered on a membrane with pores of 0.22 μm (Miipirask 40) at a flow rate of 10 ml/min. The filtered ROMIAN is stored at 2/48"C for no more than 4 days.

RBOMIAN-TT conjugates

The conditions of the process were as follows:

The initial RoMidn concentration is 2 mg/ml in a 0.2 M mass solution, the initial TT/RZMIAN ratio is 2.5/1 (m/m), the ESAS concentration is 0.25 mg/ml RB5 and the TT concentration is 10 mg/ml in a 0.2 M mass solution. 1g of ROMAN diluted in 0.2M Masi solution to obtain a final P5 concentration of 2 mg/ml (dosage of uronic acid). The purified TT solution was diluted in 0.2M Massey solution to reach a concentration of 10 mg/ml.

TT was added to the ROMOAN solution in order to achieve a final ratio of 2.5 mg TT/mg P5. 1M solution

NSI pH is brought to 5.0 x 0.05. Then ЕААС solution (7.5 mg/ml in 0.1 M Ttiz pH 7.5) was added (after 10 minutes with a peristaltic pump) to achieve 0.25 mg ЕОАС/ mg РОМідн. The resulting solution was incubated for 50 min at 257C with shaking and pH adjustment to obtain a final total time of 60 min.

Then the solution was neutralized by adding a 1M solution of Tgiz-HCI pH 7.5 and left for 30 min at 2570.

The conjugate was transferred at 4"C and left overnight with gentle shaking until the chromatography stage.

Cleaning

Before elution on Sephacryl 540O0NE, the conjugate was purified (made transparent) using a 10 μm Kieeprak filter. The flow rate was fixed at 100 ml/min. Then the conjugate was injected onto Sephakryl 5400ONE and the selection pool was evaluated by Ka value. The following criterion was used to select the pool: from 00-0.05 at 280 nm collected at the start and finish, when Ka: 0.22.

Sterilizing filtration

Prior to filtration, the bulk was brought back to room temperature. Then the conjugate was filtered on

Orisar 4" sterilizing membrane. The flow rate was fixed at 30 ml/min.

Analytical

The resulting conjugate had a final TT/P5 ratio (m/m) of 2.44/1, P5 content of 3.7956, and "RBZA/cP5 antigenicity" of 5895.

Example 14 - preparation of other polysaccharide conjugates

Covalent binding of Naetorpyiz ip'ishep gaye (Nir) PEP polysaccharide to TT was carried out by chemical condensation developed by Spy et al (Iptesiiop apa Ittipiu 1983, 40 (1); 245-256). NILB PEP polysaccharide was activated by adding SMVg and incubation at pH 10.5 for 6 minutes. Then the pH was lowered to pH 8.75 and adipic acid hydrazide (AOH) was added and the incubation was continued for another 90 minutes.

Activated PEP was attached to purified tetanus antitoxin via carbodiimide condensation using 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (ECAC). EOAS was added to activated PEP to achieve a final ratio of 0.6 mg of EPA/mg activated PEP. The pH was adjusted to 5.0 and purified tetanus antitoxin was added to achieve 2 mg TT/mg activated PEP. The resulting solution was left for three days with gentle stirring. After filtration through a 0.45 μm membrane, the conjugate was purified on a column with Sepacryl 5500NE (Rpagtasia, Zuuedep) balanced in 0.2 M mass.

Meps -TT conjugates were produced, using native polysaccharides (more than 150KkOa as measured

MAG 5), or slightly microfluidized. MepA-TT conjugates were produced using either native polysaccharides or slightly microfluidized polysaccharides greater than b0KkOa as measured

MAG 5 by the method of example 2. Mepum and Mepu-TT conjugates were produced using fractionated polysaccharides of about 100-200 kOa as measured by MAGI/5Z (see example 2). Fractionation was carried out by microfluidization using a 0-50 homogenizer. Then the polysaccharides were filtered through a 0.2 μm filter.

Activation and direct connection were performed as described in M/O96/29094 and UMO 00/56360. Briefly, the polysaccharide with a concentration of 10-20mg/ml in 2M mass, pH 5.5-6.0, was mixed with SOAR solution (100mg/ml freshly prepared in acetonitrile/PiI, 50/50) to the final

SOAR/polysaccharide ratios of 0.75/1 or 1.5/1. After 1.5 minutes, the pH was raised with sodium hydroxide solution to pH 10.0. After three minutes, tetanus antitoxin was added to achieve a protein/polysaccharide ratio of 1.51 for MepmuU, 1.2/1 for Mep, 1.5/1 for MepA, or p 1.5/1 for Meps. The reaction lasted from one to two hours.

After the coupling stage, glycine was added to a final ratio of glycine/RZ (m/m) of 7.5/1 and the pH was adjusted to 9.0. The mixture was left for 30 minutes. The conjugate was purified using a 10 μm Kieeprak filter, and then loaded into a Sephacryl column 5400HE using a buffer of 150 mM Mass, 10 mM or 5 mM Ttiz pH 7.5 for elution.

Clinical series were filtered on a sterilizing membrane Orisar 4. The resulting conjugates had an average polysaccharide:protein ratio of 1:1-1:5 (m/m).

Example 2 - determination of molecular weight using MAG 5

The detectors were connected to the RKHT column with the exclusion of the particle size from which the eluate was obtained. On the one hand, a laser light scattering detector measured the intensity of light scattered at 16 angles by a macromolecular solution, and on the other hand, an interference refractometer, placed linearly, allowed to determine the amount of washed sample. The size and shape of macromolecules in the solution can be determined by these intensities.

The average molecular weight (Mw) was determined as the sum of the masses of all varieties multiplied by their respective molecular weights and divided by the sum of the masses of all varieties. 2) ServdMmMmass molecular weight: -Miu-

TO UM pi vu Average" numerical molecular weight: -Mp-

OO UM Here (in) ! And ius: -Vm- hmm - and for:

With root mean square radius: -Am/- and Kam is the square of the radius defined by

Kht ("ti is the mass of the scattering center and ta -pi- is the distance between the scattering center and the center of gravity of the macromolecule). a) Polydispersity is defined as the ratio -Mumu/Mp-.

Meningococcal polysaccharides were analyzed by the MAG 5 method, loading into two RHCVT columns (T5KOb6000 and

BO0ORMIUKHI), which were used in combination. 25 μl of polysaccharide was added to the column and eluted with 0.75 ml of filtered water. Polysaccharides were detected using a light scattering detector (UUai Yuamlp

O5R, equipped with a 10 mW argon laser with a wavelength of 488 nm) and a low-metric refractometer (Muan OShar O5R, equipped with a P100 cell and a red X-498 nm filter).

Molecular masses of polysaccharides and recoveries of all samples were calculated by the Oerue method using polynomial fitting of order 1 in the Aviga 4.72 program.

Example C - Clinical trials to establish the effect of the linker in MepA in MepASUUM conjugate vaccine

One dose of different compositions of MepASUUU vaccine was administered to adolescents aged 15-19 years in 5 groups of 25 people in 1:1:1:1:1 random samples. Tested on the compositions were:

E1 - MepASM/U conjugated to tetanus toxoid with MepA conjugate containing AN (AON) spacer (made according to example 1) - 5/5/5/5 μg

EP2 - MepASMum conjugated to tetanus toxoid with MepA conjugate containing AN spacer (made according to example 1) - 2.5/5/2.5/2.5 μg

EZ - MepASMUU conjugated to tetanus toxoid with MepA conjugate containing AN spacer (made according to example 1) - 5/5/2.5/2.5 μg

EP4 - MepASMUU conjugated to tetanus toxoid with no spacer in any conjugate - 5/5/5/5 μg

Control group - Mepsemakh"m DSUUU

On the 30th day after vaccination, a blood sample was taken from the patients.

Blood samples were used to establish the effect of the percentage ratio of 5VA-MepA, ZVA-

MepsS, 5VA-Mepum135 and ZVA-Mepu responses one month after administration of the vaccine dose. Vaccine response was defined as 1) for initial seronegative subjects - a post-vaccination antibody titer of 21/32 in 1 month or 2) for initial seronegative subjects - an antibody titer » 4 times the pre-vaccination antibody titer.

The results

As shown in the Table below, the use of a spacer in the MepA conjugate leads to an increase in the immune response against MepA. The percentage ratio of responses increases from 6b9Uo to 90-9595 when the AN spacer was added. This was reflected in the growth in ZVA MT from 4,335 to 10,000 and growth in SMS from 5 to 20-40.

Unexpectedly, the use of AN spacer also leads to an increase in the immune response against Meps as tested by an increase in the percentage of responses and an increase in 5VA CMT. An increase could also be seen in ZVA-OMT against Mepu (6742-7122) and against Mepum (4621-5418) when the spacer was introduced. -

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Example 4 - Clinical trials to establish the effect of the linker in MepA and MepC conjugates in MepASUu/in the vaccine conjugate

One dose of different compositions of MepASUUU vaccine was administered to teenagers aged 15-19 years in 5 groups of 25 people in 1:1:1:1:1 random samples. Tested on the compositions were:

E1 - MepASMUU conjugated to tetanus toxoid with MepA and Meps conjugates containing AN spacer (made according to example 1) - 2.5/2.5/2.5/2.5 μg

E2 - MepASMUU conjugated to tetanus toxoid with MepA and Meps conjugates containing AN spacer (made according to example 1) - 5/5/2.5/2.5 μg

EZ - MepASMUU conjugated to tetanus toxoid with MepA and Meps conjugates containing AN spacer (made according to example 1) - 5/5/5/5 μg 4 - MepASM/M conjugated to tetanus toxoid with MepA conjugates containing AN spacer (made according to example 1) - 5/5/5/5 μg

Control group - Mepsemakh"m DSUUU

On the 30th day after vaccination, a blood sample was taken from the patients.

Blood samples were used to determine the effect of the percentage ratio ZVA-MepA, 5V A-

MepsS, 5VA-Mepum135 and ZVA-Mepu responses one month after administration of the vaccine dose. Vaccine response was defined as 1) for initial seronegative subjects - a post-vaccination antibody titer of 2" 1/32 in 1 month or 2) for initial seronegative subjects - an antibody titer of » 4 times the pre-vaccination antibody titer.

The results

The introduction of AN spacer into the Meps conjugate leads to an increase in the immune response against Meps as shown in the table below. This is demonstrated by the growth in the 5VA MT from 1943 to 4329 and the growth in the apii-RZS of the OMS from 7.65 to 13.13. Strong immune responses against MepA, Mepuu and Mepu were maintained. o, t. now reviews of mcg/ml of EHIZA, now reviews of mcg/ml of EHIZA

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